1. Field of the Invention
This invention relates to the medical arts. In particular, it relates to an in vitro method of isolating and expanding a cellular population enriched for cells having one or more physiological and/or immunological features of endothelial precursor cells (EPCs) and uses for those EPCs.
2. Discussion of the Related Art
Angiogenesis is a highly regulated biological process of sprouting new blood vessels from preexisting blood vessels, which supports growth and maturation. Angiogenesis begins in the mammalian embryo when primitive blood vessels are formed from endothelial cell precursors (EPCs; also known as endothelial progenitor cells or endothelial stem cells). Increasingly complex networks of vessels are formed from these primitive precursors.
Endothelial cell precursors progress through various stages before becoming mature cells. The earlier precursor cells are able to give rise to more cell types if given the right signals and environment. Cells derived from older and more mature precursors have a more limited repertoire.
Generally, molecular markers are used to keep track of the different developmental steps taken of endthelial cells, as certain molecules are present only during certain periods. Cells that can give rise to endothelial cells as well as other cell types are called hematopoietic stem/progenitor cells (HSPC) and/or hemangioblasts. The most primitive are usually derived from umbilical cord blood, peripheral blood or bone marrow and are generally positive for CD34 while being negative for CD38 and HLA-DR. CD34+ cells can be further subdivided according to their expression of CD45RA and CD71. Cells which are CD34+ CD45RA− and CD71− give rise to multipotent progenitor cells, including those which will produce endothelial cell precursors (angioblasts); those that are CD34+ CD45RA+ and CD71−, give rise to granulocyte and monocye progenitors whereas those which are CD34+ CD45RA− and CD71+ give rise to erythrocyte progenitors. (Mayani H and Lansdorp P M, Biology of Human Umbilical Cord Blood-Derived Hematopoietic Stem/Progenitor Cells. Stem Cells. 1998; 16:153–165).
CD34+ cells can be manipulated by the right culture conditions to produce mature endothelial cells expressing von Willebrand factor, CD31, CD54 and CD62. (Mayani H and Lansdorp P M, Biology of Human Umbilical Cord Blood-Derived Hematopoietic Stem/Progenitor Cells. Stem Cells. 1998; 16:153–165). Considerable controversy exists regarding the specific pathway(s), as well as the number of intermediates in the transition between hemangioblasts and differentiated endothelial cells. However, “presumptive hemangioblasts” express CD34, FLK-1, SCL, LMO2 and GATA-2. (Orkin S H and Zon L I. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nature Immunology. 2002. 3(4):323–328).
Even though there is strong support for the expression of CD34 in hematopoietic stem cells, many groups have reported it to be “reversible”, “changing”, or “absent”. (Ishikawa F. Reversible expression of CD34 by hematopoietic stem cells. Ronsho Ketsueki. 2002. 43(1):1–4; Ogawa M. Changing phenotypes of hematopoietic stem cells. Exp Hematol. 2002. 30(1):3–6; Huss R. Perspectives on the morphology and biology of CD34-negative stem cells. J Hematother Stem Cell Res. 2000. 9:783–793). These controversies may be due to the different origins of these stem cells (organs vs. blood or bone marrow) or to the analysis of a vast number of potential intermediates in the differentiation pathway.
There is a specific set of markers present only in EPCs, but not in differentiated endothelial cells. These include AC133, CD166, and AML-1. (Asahara T., et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997. 275:964–967; Matsumoto K., et al. In vitro proliferation potential of AC133 positive cells in peripheral blood. Stem Cells. 2000. 18:196–203; Ohneda O., et al. ALCAM (CD166): its role in hematopoietic and endothelial development. Blood. 2001. 98(7): 2134–2142; Okuda T., et al. RUNX1/AML-1: a central player in hematopoiesis. Int. J. Hematol. 2001. 74:252–257).
In contrast, some markers are expressed only on “mature endothelial cells”, and not the EPCs, such as CD31, CD36 and CD62, V-Cadherin. (Reyes M., et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. 2002. 109(3):337–346).
When precursor cells divide, they can either produce daughter cells more mature than themselves (proliferation) or produce more precursor cells like themselves (expansion). The cellular environment, including cytokines and growth factors, controls these processes. (Mayani H and Lansdorp P M, Biology of Human Umbilical Cord Blood-Derived Hematopoietic Stem/Progenitor Cells. Stem Cells. 1998; 16:153–165).
In adults, nonpathogenic angiogenesis is restricted and transient, for example, as part of the wound healing process and during the female reproductive cycle in the endometrium and ovarian follicle.
Because of the role angiogenesis is thought to play in human diseases, pathogenic angiogenesis has been intensively studied. The highly regulated process of angiogenesis is considered a physiological response to the balance between the actions of proangiogenic and antiangiogenic factors, synthesized by endothelial cells, stromal cells, blood, the extracellular matrix, and tumor cells (Carmeliet, P. and Jain, R. K., Angiogenesis in cancer and other diseases, Nature (2000) 407:249–257 [2000]). When proangiogenic factors are synthesized, stimulated by metabolic stress, mechanical stress, inflammation, or genetic mutations, new blood vessels are created from preexisting ones and pathogenic states result (Carmeliet, P. and Jain, R. K. [2000]). Proangiogenic factors create new blood vessels in six distinct steps: vascular destabilization caused by pericyte detachment, extracellular matrix degradation by endothelial proteases, endothelial cell migration, endothelial cell proliferation, tube formation by endothelial cells, and recruitment of pericytes to stabilize vasculature. (Sato, Y., Molecular mechanism of angiogenesis. Transcription factors and their therapeutic relevance, Pharm & Ther 87:51–60 [2000]).
All of these steps are mediated by proangiogenic factors acting in concert with one another. For example, vascular endothelial growth factor (VEGF) and related molecules stimulate vessel leakage, matrix metalloproteases (MMPs) remodel extracellular matrix and release and activate growth factors, platelet-derived growth factor BB (PDGF-BB) and receptors recruit smooth muscle cells, vascular endothelial growth factor receptor (VEGFR) and NRP-1 integrate angiogenic and survival signals, plasminogen activator inhibitor-1 (PAI-1) stabilizes nascent vessels, and angiopoietin 1 (Ang1) and its receptor precursor (Tie2) in turn stabilize vessels (Carmeliet, P. and Jain, R. K. [2000]) 407:249–257).
For many years, tube formation on a reconstituted basement membrane matrix (MATRIGEL™ biological cell culture substrate) has been the assay of choice for the assessment of angiogenesis in vitro (Baatout S., Endothelial differentiation using Matrigel (review), Anticancer Res. 17: 451–6 [1997]; Benelli, R. and Albini, A., In vitro models of angiogenesis: the use of Matrigel, Int. J. Biol. Markers. 14, 243–6 [1999]). MATRIGEL™ biological cell culture substrate is derived from the extracellular matrix deposited by a mouse EHS tumor. MATRIGEL™ biological cell culture substrate is a complex mixture of basement membrane proteins (laminin, type IV collagen, entactin/nidogen, and heparan sulfate proteoglycans) and it also contains certain growth factors. (Kleinman, H. K. et al., Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma, Biochemistry. 21: 6188–93 [1982]; Vukicevic, S. et al., Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components, Exp Cell Res. 202: 1–8 [1992]). Unlike the commonly used type I collagen gels (Schor, A. M. et al., Collagen gel assay for angiogenesis. In: Methods in molecular medicine, angiogenesis protocols [Murray J. C., ed.], Humana Press, Totowa, N.J., pp. 145–62 [2001]), the reconstituted basement membrane matrix contains natural substrates that endothelial cells encounter under physiological conditions, i.e., basement membrane components. It has previously been thought that endothelial cells plated on a reconstituted basement membrane matrix 1) stop proliferating, 2) migrate and form capillary-like tubes by 24–36 hr, 3) do not invade the matrix, 4) collapse into clumps, and 5) die. (E.g., Pollman et al., Endothelial cell apoptosis in capillary network remodeling., J. Cell Physiol. 178, 359–70 [1999]; Benelli and Albini [1999]). Because this was thought to be the endpoint of the assay, no previous experiments have extended beyond this point.
The survival of tumors is now considered to be dependent upon tumor angiogenesis. For this reason, cancer chemotherapy is beginning to exploit angiogenesis inhibition as a mechanism to limit tumor metastases and angiogenesis is increasingly being used as a diagnostic/prognostic marker. For example, tumor vascularity in solid tumors may inversely correlate with prognosis, and both basic fibroblast growth factor (bFGF; or FGF-2) and VEGF expression have been reported to predict prognosis. (Takahashi, Y. et al., Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer, Cancer Res 55:3964–68 [1995]). Breast cancer prognosis can also be based on the extent of angiogenesis. (Weidner, N. et al., Tumor angiogenesis: a new significant and independent prognostic factor in early-stage breast carcinoma, J. Natl. Cancer Inst. 84:1875–1887 [1992]; Horak, E. R. et al., Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metasteses and survival in breast cancer, Lancet 340:1120–1124 [1992]). Not only are tumor growth, progression, and metastasis dependent on access to vasculature, but it is also apparent that an “angiogenic switch” is activated during the transition from mid to late dysplasia, causing a change in tissue angiogenic phenotype preceding the histological tissue transition. (Hanahan, D. and Folkman, J., Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 86:353–64 [1996]).
During tumor-associated angiogenesis, sustained production of angiogenic factors by cancer cells, or indirect macrophage stimulation, causes dysregulated immature vessel growth. (Folkman, J. and Shing, Y., Angiogenesis, J Biol. Chem. 267:10931–10934[1992]). Several cytokines and growth factors are highly associated with intratumoral angiogenesis, including bFGF and VEGF which modulate angiogenesis in vivo with a paracrine mode of action. (Bikfalvi, A. et al., Biological roles of fibroblast growth factor-2, Endocr. Rev. 18:26–45 [1997]; Ferrara, N. and Davis-Smyth, T., The biology of vascular endothelial growth factor, Endocr Rev 18:4–25 [1997]; Relf, M et al., Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis, Cancer Res. 57(5):963–69 [1997]; Linderholm, B. et al., Vascular endothelial growth factor is of high prognostic value in node-negative breast carcinoma, J. Clin. Oncol. 16:3121–28 [1998]). bFGF and VEGF may synergistically influence angiogenesis, with bFGF modulating endothelial expression of VEGF through both autocrine and paracrine actions. (Seghezzi, G. et al., Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: An autocrine mechanism contributing to angiogenesis, J. Cell. Biol. 141(7):1659–73 [1998]).
For these reasons, drugs acting through an antiangiogenic mechanism are contemplated to prevent neoplastic growth. As an example, Hunter et al. described a method of treating a tumor excision site with a composition including paclitaxel or a paclitaxel analog with a polymer to prevent residual blood vessel formation. (U.S. Pat. No. 5,886,026).
In addition to cancer, other pathological states require angiogenesis including diabetes mellitus, Alzheimer's disease, asthma, and hypertension. The pathological progression in endometriosis is also thought to involve angiogensis. (E.g., Taylor, R N et al., Angiogenic factors in endometriosis, Ann N Y Acad Sci 955:89–100 [2002]; Shawki, O et al., Apoptosis and angiogenesis in endometriosis: relationship to development and progression, Fertil Steril. 77 Suppl 1:S44 [2002]; Gazvani, R et al., Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis, Reproduction 123(2):217–26 [2002]; Taylor, R N et al., Endocrine and paracrine regulation of endometrial angiogenesis, Ann N Y Acad. Sci. 943:109–21 [2001]; Gazvani, R et al., New considerations for the pathogenesis of endometriosis, Int J Gynaecol Obstet. 2002 Feb.;76(2):117–26 [2002]; Fujimoto, J et al., Angiogenesis in endometriosis and angiogenic factors, Gynecol Obstet Invest. 48 Suppl 1:14–20 [1999]; Healy, D L et al., Angiogenesis: a new theory for endometriosis, Hum. Reprod. Update. 1998 Sep.–Oct.;4(5):736–40 [1998]; Matsuzaki, S et al., Angiogenesis in endometriosis, Gynecol. Obstet. Invest. 46(2):111–15 [1998]).
Inflammatory disorders can involve excessive angiogenesis in various organs. Blood cells including platelets, mast cells, monocytes, and macrophages release angiogenic factors, such as VEGF, ANG1, bFGF, TGF-β1, PDGF, TNF-α, hepatocyte growth factor (HGF), and insulin-like growth factor (IGF-I). Additionally, blood cells contain proteases that degrade barriers for migrating vasculature and activate growth factors from extracellular matrix. Wound repair is an example of how the inflammatory response influences angiogenesis in a non-pathogenic way. Angiogenesis in wound repair can be described in the following steps: 1) endothelial cells are released from the basement membrane degraded by metalloproteinases and other proteases, and 2) the endothelial cells migrate to connective tissue and differentiate into tubes where they resynthesize the basement membrane, all in response to the proangiogenic factors being secreted at the wound site. (Kleinman, H. K. and Malinda K. M., Role of angiogenesis in wound healing, in Angiogenesis Inhibitors and Stimulators: Potential Therapeutic Implications, Ed. Mousa, S. A., pp. 102–109 [2000]).
The primary cause of pathological angiogenesis in non-neoplastic disease states is hypoxia. Hypoxia-induced transcription factors (HIFs) induce the expression of angiogenic factors including VEGF, nitric oxide synthase, PDGF, Ang2, and others (Carmeliet, P. and Jain, R. K. [2000]). As a result, hypoxia-induced angiogenesis leads to blindness in premature newborns, diabetics, and hemorrhagic rupture of atherosclerotic plaques. Additionally, vascular remodeling caused by hypoxia induces chronic obstructive lung disease, characterized by the thickening of vascular muscular coat and pulmonary hypertension. Although hypoxia-induced angiogenesis can be pathological, it also salvages ischemic myocardium and promotes survival after stroke. For these reasons, the use of proangiogenic factors has been proposed as therapy for ischemic diseases, such as arteriosclerotic occlusion of the lower limb or angina pectoris/myocardial infarction.
Diabetic retinopathy, the most severe ocular complication of diabetes mellitus, may be defined as a disease of retinal microvasculature. Diabetic retinopathy is the leading cause of new blindness in persons 25 to 74 years of age in the United States, accounting for about 8,000 new blindness cases each year. (Aiello L P et al., Diabetic retinopathy, Diabetes Care 21:143–156 [1998]; Lim J I et al., Review of diabetic retinopathy, Curr. Opin. Ophthalmol. 2:315–323 [1991]). Two types of diabetic retinopathy are recognized clinically: (1) nonproliferative diabetic retinopathy (NPDR), associated with retinal ischemia, pericyte loss, capillary closure, retinal infarctions/cotton wool spots, retinal hemorrhages, microaneurisms, intraretinal microvascular abnormalities, and macular edema; and (2) proliferative diabetic retinopathy (PDR), associated with intravitreal hemorrhages, optic disc or peripheral neovascularization, preretinal fibrovascular membranes, and vitreoretinal traction with retinal detachments (Aiello L P et al. [1998]; Lim J I et al. [1991]). Sadly, 43% of juvenile-onset and 60% of adult-onset diabetics lose vision within 5 years of the onset of PDR.
Supporting the conclusion that diabetic retinopathy is a disease of retinal microvasculature, abnormally high concentrations of angiogenic growth factors have been detected in the vitreous of diabetic retinopathy and PDR patients. (Aiello L P, and Hata Y., Molecular mechanisms of growth factor action in diabetic retinopathy, Curr. Opin. Endocrinol. Diabetes 6:146–156 [1999]; Boulton, M. et al., Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management, Br. J. Ophthalmol. 81:228–233 [1997]; Freyberger, H. et al., Increased levels of platelet-derived growth factor in vitreous fluid of patients with proliferative diabetic retinopathy, Exp. Clin. Endocrinol. Diabetes 108:106–109 [2000]). Additionally, VEGF induced by hypoxia and hyperglycemia has been implicated in causing PDR neovascularization and vascular hyperpermeability. (Aiello L P, and Hata Y., Molecular mechanisms of growth factor action in diabetic retinopathy, Curr. Opin. Endocrinol. Diabetes 6:146–156 [1999]; Aiello, L P and Wong, J S, Role of vascular endothelial growth factor in diabetic vascular complications, Kidney Int. 58 (Suppl. 77):113–119 [2000]).
Retinas in proliferative diabetic retinopathy (PDR) have increased expression of VEGF, PlGF, and tenascin, a vascular basement membrane protein. (E.g., Ljubimov A V et al., Basement membrane abnormalities in human eyes with diabetic retinopathy, J. Histochem. Cytochem. 1996;44:1469–1479 [1996]; Spirin K S et al., Basement membrane and growth factor gene expression in normal and diabetic human retinas, Curr. Eye Res. 18:490–499 [1999]). Hypoxia-inducible VEGF is considered as the main growth factor that mediates PDR neovascularization (Smith L E et al., Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor, Nat. Med. 5:1390–1395 [1999]).
However, VEGF inhibitors only partially prevent ocular neovascularization and vessel hyperpermeability. (Campochiaro, P A, Retinal and choroidal neovascularization, J. Cell Physiol. 184:301–310 [2000]; Aiello L P, Vascular endothelial growth factor. 20th-century mechanisms, 21st-century therapies. Invest. Ophthalmol. Vis. Sci. 38:1647–1652 [1997]; Ozaki H et al., Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization, Am. J. Pathol. 156:697–707 [2000]; Aiello L P, Vascular endothelial growth factor and the eye: Biochemical mechanisms of action and implications for novel therapies, Ophthalmic Res. 1997;29:354–362; Aiello L P et al., Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective β-isoform-selective inhibitor, Diabetes 46:1473–1480 [1997]; Campochiaro P A, Retinal and choroidal neovascularization, J. Cell Physiol. 184:301–310 [2000]; Penn J S, Bullard L E, VEGF signal transduction proteins ERK-1 and ERK-2 are targets for the inhibition of retinal angiogenesis, Exp. Eye Res. (ICER Abstracts) 71(Suppl. 1):S.5 [2000]).
This implies that other factors may be involved in this process. (See, Castellon, R. et al., Effects of Angiogenic Growth Factor Combinations on Retinal Endothelial Cells, Exp. Eye Res. 74:523–35 [2002]). Growth factor synergies have been reported in other tissues. (Goto F et al., Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels, Lab. Invest. 69:508–517 [1993]; Stavri G T et al., Hypoxia and platelet-derived growth factor-BB synergistically upregulate the expression of vascular endothelial growth factor in vascular smooth muscle cells, FEBS Lett. 358:311–315 [1995a]; Stavri G T et al., Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia, Circulation 92:11–14 [1995b]; Hata Y et al., Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway, Diabetes 48:1145–1155 [1999]; Miele C et al., Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways, J. Biol. Chem. 275:21695–21702 [2000]).
There remains a need for an in vitro method of isolating and expanding a cellular population enriched for endothelial precursor cells that can be used to further the study of biochemical mechanisms of angiogenesis and antiangiogenesis, and can be employed to screen substances for potential new proangiogenic and antiangiogenic agents that could be useful for therapeutic purposes. These and other benefits are provided by the present invention as described herein.